Several hundred naturally occurring, monoterpenes are known, and essentially all are biosynthesized from geranyl pyrophosphate, the ubiquitous C.sub.10 intermediate of the isoprenoid pathway (Croteau and Cane, Methods of Enzymology 110:383-405 1985!; Croteau, Chem. Rev. 87:929-954 1987!). Monoterpene synthases, often referred to as "cyclases," catalyze the reactions by which geranyl pyrophosphate is cyclized to the various monoterpene carbon skeletons. These enzymes have received considerable recent attention because the cyclization process determines the basic character of the monoterpene end-products and because the cyclization mechanism is quite complex, involving multiple steps in which many of the carbon atoms of the substrate undergo alterations in bonding, hybridization, and configuration (Croteau and Cane, Methods of Enzymology 110:383-405 1985!; Croteau, Chem. Rev. 87:929-954 1987!). Research on monoterpene cyclases has also been stimulated by the possible regulatory importance of these enzymes that function at a branch point in isoprenoid metabolism (Gershenzon and Croteau, Biochemistry of the Mevalonic Acid Pathway to Terpenoids (Towers and Stafford, eds.), Plenum Press, New York, N.Y., pp. 99-160 1990!) as well as by the commercial significance of the essential oils (Guenther, The Essential Oils, Vols. III-VI (reprinted) R. E. Krieger, Huntington, N.Y. 1972!) and aromatic resins (Zinkel and Russell, Naval Stores: Production, Chemistry, Utilization, Pulp Chemicals Association, New York 1989!) and the ecological roles of these terpenoid secretions, especially in plant defense (Gershenzon and Croteau, in "Herbivores: Their Interactions with Secondary Plant Metabolites," Vol. I, 2nd Ed. (Rosenthal and Berenbaum, eds.) Academic Press, San Diego, Calif., pp. 165-219 1991!; Harbome, in "Ecological Chemistry and Biochemistry of Plant Terpenoids," (Harborne and Tomas-Barberan, eds.) Clarendon Press, Oxford, Mass., pp. 399-426 1991!).
One of the major classes of plant monoterpenes is the monocyclic p-menthane (1-methyl-4-isopropylcyclohexane) type, found in abundance in members of the mint (Mentha) family. The biosynthesis of p-menthane monoterpenes in Mentha species, including the characteristic components of the essential oil of peppermint (i.e., (-)-menthol) and the essential oil of spearmint (i.e., (-)-carvone), proceeds from geranyl pyrophosphate via the cyclic olefin (-)-limonene (Croteau, Planta Med. 57 (suppl): 10-14 1991!), as shown in FIG. 1. The transformation of geranyl pyrophosphate to limonene is seemingly the least complicated terpenoid cyclization (Croteau and Satterwhite, J. Biol. Chem. 264:15309-15315 1989!) in having ample precedent in solvolytic model studies (Cramer and Rittersdorf, Tetrahedron 23:3015-3022 1967!; Haley et al., J. Chem. Soc. C., pp. 264-268 1969!; Kobayashi et al., Chem. Lett., pp. 1137-1138 1976!; Vial et al., Tetrahedron 37:2351-2357 1981!), and the responsible enzyme has become a prototype for the terpenoid cyclization reaction (Cori, Phytochemistry 22:331-341 1983!; Pauly et al., Plant Cell Rep. 5:19-22 1984!; Suga et al., Chem. Lett., pp. 115-118 1988!; Perez et al., Plant Physiol. Biochem. 28:221-229 1990!; Rajaonarivony et al., Arch. Biochem. Biophys. 299:77-82 1992!). The enzyme that produces the (-)-4S-enantiomer (geranyl pyrophosphate:(-)-4S-limonene cyclase or, simply, (-)-4S-limonene synthase) has been purified from peppermint (Mentha x piperita) and spearmint (Mentha spicata) oil glands (Alonso et al., J. Biol. Chem. 267:7582-7587 1992!), and highly specific antibodies directed against this enzyme have been prepared (Alonso et al., Arch. Biochem. Biophys. 301:58-63 1993!). In properties and mechanism of action (Rajaonarivony et al., Arch. Biochem. Biophys. 299:77-82 1992!; Rajaonarivony et al., Arch. Biochem. Biophys. 296:49-57 1992!), it seemingly catalyzes a slow, possibly rate-limiting, step of monoterpene biosynthesis in Mentha (Gershenzon and Croteau, Biochemistry of the Mevalonic Acid Pathway to Terpenoids (Towers and Stafford, eds.), Plenum Press, New York, N.Y., pp. 99-160 1990!).
A detailed understanding of the control of monoterpene biosynthesis and of the cyclase reaction mechanism requires the relevant cDNA clones as tools for evaluating patterns of developmental and environmental regulation and for examining active site structure function relationships. There have been no previous reports of the cloning of a monoterpene cyclase although the molecular cloning of a sesquiterpene cyclase of plant origin (epi-aristolochene synthase from tobacco) has recently been reported (Facchini and Chappell, Proc. Natl. Acad. Sci. USA 89:11088-11092 1992!), and the cloning of a diterpene cyclase (casbene synthase from castor bean) has also been accomplished.